This proposed research will further our understanding of the mode of action of colicins through characterization of altered structural and functional properties of both the interacted colicin molecules and the bacterial cells. We shall use radioactive colicin of high specific radioactivity, labeled with H3 or C14 amino acids to treat colicin sensitive and tolerant bacteria. Mechanical disruption of the treated bacteria, ultracentrifugation, and sucrose gradients will be employed to obtain and examine the association of labeled colicin component with soluble components, cell wall and cytoplasmic membranes. We shall investigate modifications in the structure of the interacted labeled colicin molecules utilizing gel electrophoresis and peptide analysis studies. From the standpoint of the interacted bacterial cells, in vivo and in vitro studies of the effects of colicin K on composition and bioenergetics of membranes are proposed and should lead to the identification of the primary defects caused by the colicin. These studies should also shed light on the details of oxidative phosphorylation in bacteria and the nature of ATP generated energized state of bacterial membranes. To study the effects of colicin K on composition and bioenergetic functions of cytoplasmic membranes, lipid analysis methodology, gel electrophoresis studies, and spectrophometric assay of the ATP linked reverse electron transfer reactions will be used. Double label techniques and various biochemical methods will be used to purify and characterize the structure and function of solubilized factors from bacterial cells. These factors are required for the ATP linked reactions and appear impaired in the colicin-treated bacteria. Establishment of an in vitro effect of colcin on energy linked functions of bacterial membranes allows similar investigations on eucaryotic membranes, which may prove to be of significance in evaluating the potential pathogenicity of colicin molecules.